Electron spin resonance spectroscopy (ESR) is a highly efficient technique able to access a wide range of information about the unfavourable effects caused by a chemical or a drug. ESR in spin labelling ... [more ▼]

Electron spin resonance spectroscopy (ESR) is a highly efficient technique able to access a wide range of information about the unfavourable effects caused by a chemical or a drug. ESR in spin labelling fits well in with the study of membranes and particularly with the changes in lipid bilayer organisation induced by drug. Our team previously developed a way to quantify the effective lipid bilayer microviscosity of cell membranes and consequently put in evidence the fluidity effect of the propofol. Recently, the importance of lipid raft domains has been shown due to their important role as a platform for signal transduction and protein sorting. We propose to highlight the effect of the Randomly methylated beta cyclodextrin (Rameb) on these domains on membrane model (liposomes) as well as on colon carcinoma cell line (HCT-116). Futhermore, ESR in spin trapping is used in order to identify and quantify the generation of Reactive Oxygen Species (ROS) in cells. An ESR study on human colon carcinoma cell line has highlighted the cytotoxicity of the photosensitizer pyrophephorbide-a methyl ester. Using an intracellular located spin trap (4-pyridyl 1-oxide-N-tert-butylnitrone, POBN), it has been shown that the photoexcitation of the dye is able to generate superoxide anions, hydroxyl radicals and singlet oxygen. Moreover, ESR is one of the most sensitive method for measuring cellular oxygen consumption. Our team has studied the alterations of oxygen respiratory in human tubular renal cells treated with an endotoxin (lipopolysaccharide, LPS). The incubation of HK-2 cells with LPS elicited a decreased in oxygen consumption suggesting a down-regulation of the cells metabolism. [less ▲]

A new way to study the action of cyclodextrin was developed to quantify the damage caused on cell membrane and lipid bilayer. The Electron Spin Resonance (ESR) spectroscopy was used to study the action of ... [more ▼]

A new way to study the action of cyclodextrin was developed to quantify the damage caused on cell membrane and lipid bilayer. The Electron Spin Resonance (ESR) spectroscopy was used to study the action of Randomly methylated-beta-cyclodextrin (Rameb) on living cells (HCT-116). The relative anisotropy observed in ESR spectrum of nitroxide spin probe (5-DSA and cholestane) is directly related to the rotational mobility of the probe,which can be further correlated with themicroviscosity. The use of ESR probes clearly shows a close correlation between cholesterol contained in cells and cellular membrane microviscosity. This study also demonstrates the Rameb ability to extract cholesterol and phospholipids in time- and dose-dependent ways. In addition, ESR spectra enabled to establish that cholesterol is extracted from lipid rafts to form stable aggregates. The present work supports that ESR is an easy, reproducible and noninvasive technique to study the effect of cyclodextrins on cell membranes. [less ▲]

Pyropheophorbide-a methyl ester (PPME), a derivative of chlorophyll a, is a second-generation photosensitizer and is studying largely in vitro for nearly a decade on cancerous cells. It has been ... [more ▼]

Pyropheophorbide-a methyl ester (PPME), a derivative of chlorophyll a, is a second-generation photosensitizer and is studying largely in vitro for nearly a decade on cancerous cells. It has been previously established on HCT-116 (human colon carcinoma cell line) that PPME is a molecule able to create apoptotic and necrotic death (Matroule et al). The cytotoxicity of PPME is presumed to be induced by reactive oxygen species (ROS) generated by the photoexcited molecule. Actually, to the best of our knowledge, no experimental evidence enables to confirm this supposition in an indubitable manner. Electron spin resonance (ESR) associated with spin trapping technique is a powerful method to detect, quantify and identify the ROS produced after photoactivation of PPME. Previous results indicate that PPME penetrates inside cells and localizes inside specific organelles (endothelial reticulum, Golgi apparatus and lysosome) (Matroule et al). The recent researches of Guelluy et al have also clearly demonstrated the presence of PPME inside mitochondrion. Consequently, ESR experiments were performed using an intracellular located spin trap, POBN (4-pyridyl 1-oxide-N-tert-butylnitrone), in order to detect in situ the ROS production. It has been shown that PPME is able to generate superoxide anions and hydroxyl radicals. Irradiation of the dye in HCT-116 cells in the presence of POBN spin trap and ethanol scavenger (2%, a non-toxic concentration) leads to the apparition of the ESR spectrum characteristics of POBN/ethoxy adduct. To assess the extent of contribution of ROS and to determine a possible reaction mechanism, competition experiments with specific quencher agents were carried out. Addition of catalase (CAT), a hydrogen peroxide quencher, or superoxide dismutase (SOD), a superoxide anion quencher, inhibits 30% of the signal. The parallel effect of SOD and CAT suggest that superoxide anion and hydrogen peroxide are involved in the generation of hydroxyl radicals via a Fenton reaction. This assertion is reinforced by the 20% reduction of signal intensity when adding desferroxamine, a Fe3+ chelator also implicated in Fenton reaction. Addition of DABCO, a quencher of singlet oxygen, to cells reduces 70% of the POBN/ethoxy adduct signal intensity. [less ▲]

In the present work, the effect of Randomly-methylated-b-cyclodextrin (Rameb) on the microviscosity of dimyristoyl-L-a phosphatidylcholine (DMPC) bilayer was investigated using the electron spin resonance ... [more ▼]

In the present work, the effect of Randomly-methylated-b-cyclodextrin (Rameb) on the microviscosity of dimyristoyl-L-a phosphatidylcholine (DMPC) bilayer was investigated using the electron spin resonance (ESR) technique. The ability of Rameb to extract membrane cholesterol was demonstrated. For the first time, the percentage of cholesterol extracted by Rameb from cholesterol doped DMPC bilayer was monitored and quantified throughout a wide Rameb concentration range. The effect of cholesterol on the inner part of the membrane was also investigated using 16-doxyl stearic acid spin label (16-DSA). 16-DSA seems to explore two different membrane domains and report their respective microviscosities. ESR experiments also establish that the presence of 30% of cholesterol in DMPC liposomes suppresses the jump in membrane fluidity at lipids phase-transition temperature (23.9°C). [less ▲]